Lyme disease, caused by the spirochete Borrelia burgdorferi (Bb), is the most common vector-borne disease in U.S. Complement resistance, enabling bloodstream survival, is likely essential for spirochetes to disseminate to distant sites. A critical step in complement activation is the formation of C3 convertases C4b2a or C3bBb, complexes that lead to inflammation, opsonization and pathogen lysis. Potential tissue injury due to complement activation necessitates stringent control by serum complement regulatory proteins (CRPs) that bind to and promote degradation of complement proteins. To avoid complement-mediated killing, pathogens often produce surface proteins that bind CRPs, a phenomenon thought to contribute to bacteremia and colonization of tissues. For Bb, however, a rigorous demonstration of this is lacking. Bb surface proteins BBK32 and DbpA are well-studied Bb extracellular matrix (ECM) adhesins, and we found that OspC also functions as an ECM-binding adhesin. Our novel findings forming the basis for this proposal are that all three adhesins also promote serum survival in vitro and bloodstream survival in vivo. DbpA and BBK32 both bind to C4BP, which is predicted to block the formation of C4b2a. Bb producing DbpAI156A, a DbpA point mutant deficient for C4BP- but not ECM-binding, was delayed in blood and joint colonization, suggesting that DbpA mediates early stage bacteremia. OspC bound to C4b, predicted to block the formation of C4b2a, and allelic variants of OspC displayed distinct C4b-binding activities that correlated with serum resistance activity in vitro and early-stage bacteremia in vivo. These are the first demonstrations that Bb produces complement evasion factors that interfere with both the classical and lectin, and the first to demonstrate tha any complement resistance factor plays an important role in Bb dissemination in the mammalian host. However, our data also suggest that other, unknown bacterial factors may play a role in persistent infection. To determine mechanisms of complement evasion by Bb and to identify novel serum resistance factors, we will 1) Define the contribution of BBK32-mediated C4BP binding to serum resistance and bacteremia by evaluating a C4BP-binding deficient BBK32 mutant for serum resistance and bloodstream survival; 2) Determine whether C4b-binding activity is required for OspC-mediated serum resistance and early bacteremia by testing C4b-binding deficient OspC mutants for loss of ability to promote these phenotypes; 3) Characterize mechanisms by which DbpA, BBK32 and OspC block complement activation by examining whether DbpA- or BBK32- C4BP complexes promote proteolysis of C4b and inhibit bacterial lysis and whether the C4b-OspC complex inhibits the formation of C4b2a; and 4) Perform a Tn-Seq -based large-scale screen to identify Bb genes encoding factors that promote serum resistance. This work will have significant impact on potential therapeutic interventions and understanding fundamental mechanisms of B. burgdorferi pathogenesis and life cycle. .